In exciting new studies, we showed that phthalic acid esters and lipid lowering drugs (e.g., diethylhexylphthalate (DEHP) and WY-14,643) activate phagocytosis by the resident hepatic macrophages, Kupffer cells. Further, increased cell proliferation was blocked in vivo by both an antibody to TNFalpha and methyl palmitate, an inactivator of Kupffer cells. Based on this new information, we hypothesize that phthalates activate Kupffer cells to produce mitogenic cytokines that stimulate cell proliferation and cause tumors. To test this hypothesis, three questions will be posed. First, are Kupffer cells responsible for increased cell proliferation due to plasticizers? Kupffer cells will be treated with DEHP and the lipid lowering drug WY-14,643 in vitro or isolated from rats treated in vivo and cultured. Conditioned media from Kupffer cells will be added to cultured hepatocytes and cell proliferation will be assessed. Similar experiments will be performed with human macrophages and hepatocytes since whether or not these chemicals cause cancer in humans remains controversial. Nimodipine, methyl palmitate and dietary glycine will be used to prevent or minimize activation of Kupffer cells. We expect these experiments to demonstrate conclusively that TNFalpha produced by Kupffer cells is responsible for enhanced cell proliferation caused by plasticizers. Second, experiments will be performed to understand how phthalates activate Kupffer cells to produce mitogens. We propose that phthalates will enter Kupffer cells membranes based on their lipophilicity, increase intracellular calcium, and activate PKC by altering second messenger systems. Accordingly, uptake of radiolabelled drugs of different lipid solubility will be compared. Further, the effect of phthalate treatment in vivo on intracellular calcium and PKC will be measured in isolated Kupffer cells. To determine if PKC activates NADPH oxidase and increases NF/kappa/B-mediated TNFalpha production, superoxide and NF/kappa/B will be monitored and correlated with changes in TNFalpha mRNA. Third, can phthalate-stimulated increases in cell proliferation and tumors be prevented in vivo by modulation of Kupffer cells? Dietary glycine, which inhibits Kupffer cell activation, will be used to prevent cell proliferation, changes in apoptosis, and the formation of preneoplastic foci and tumors, which is the "gold-standard." By pursuing these new hypotheses, we will fill important gaps in our knowledge regarding mechanisms. Another important impact of this work will be to provide governmental regulators with mechanistic information which will allow them to shift emphasis away from the fact that these chemicals induce peroxisomes and focus on cell proliferation.